The present disclosure relates generally to capture, detection and sequencing of nucleic acids. More specifically, the disclosure relates to formation and detection of ternary complexes that each include a primed template nucleic acid, polymerase, and cognate nucleotide, for example, in a Sequencing By Binding™ (SBB™) method.
SBB™ technology employs transient binding of a polymerase and cognate nucleotide to a primed template nucleic acid as a means to identify the template base that is at the end of the primer. Serial steps of extending the primer and detecting the next template base allow the sequence of the template to be determined. Exemplary SBB™ techniques are disclosed, for example, in commonly owned U.S. Pat. App. Pubs. 2017/0022553 A1 and 2018/0044727 A1; and U.S. Pat. App. Ser. No. 15/873,343 (published as US 2018/0208983 A1) and Ser. No. 15/851,383 (published as US 2018/0187245 A1), each of which is incorporated herein by reference.
A difficulty of SBB™ technology is that a ternary complex is an equilibrium binding product. An equilibrium binding product coexists in solution with non-bound binding partners. Removal of non-bound binding partners from an equilibrium reaction causes the binding product to dissociate. When using labeled nucleotides in the SBB™ procedure, non-bound, labeled nucleotide provides a desired function of maintaining the equilibrium that, in turn, maintains the ternary complex. However, the non-bound, labeled nucleotide undesirably produces background signal that can obscure detection of the ternary complex. A similar difficulty can arise when using labeled polymerase in lieu of labeled nucleotides.
What is needed is a method to maintain detectable levels of ternary complexes while decreasing the concentration of labeled background components. The present invention satisfies this need and provides related advantages as well.